Isolated DNA sequence capable of serving as regulatory element in a chimeric gene which can be used for the transformation of plants

ABSTRACT

An isolated DNA sequence capable of serving as regulatory element in a chimeric gene which can be used for the transformation of plants is disclosed. A chimeric gene for the transformation of plants is also disclosed. The gene comprises at least, in the direction of transcription, a promoter sequence, a transgene and a regulatory element, characterized in that the regulatory element consists of at least one intron 1 in the noncoding 5′ region of a plant histone gene allowing the expression of the protein in the zones undergoing rapid growth. The production of transgenic plants is also disclosed.

[0001] The present invention relates to the use of a regulatory elementisolated from transcribed plant genes, of new chimeric genes containingthen and to their use for the transformation of plants.

[0002] Numerous phenotypic characters associated with the expression ofone or more gene elements can be integrated into the genome of plantsand thus confer on those transgenic plants advantageous agronomicproperties. In a nonexhaustive manner, there may be mentioned: theresistances to pathogenic agents for crops, the resistance to phytotoxicplant-protection products, the production of substances of dietary orpharmacological interest. In addition to the isolation andcharacterization of the gene elements encoding these various characters,an appropriate expression should be ensured. This appropriate expressionmay be situated both at the qualitative and quantitative levels. At thequalitative level, for example the spatial level: preferentialexpression in a specific tissue, or temporal level: inducibleexpression; at the quantitative level, by the accumulated quantity ofthe product of expression of the gene introduced. This appropriateexpression depends, for a large part, an the presence of regulatory geneelements associated with the transgenes, in particular as regards thequantitative and qualitative elements. Among the key elements ensuringthis appropriate regulation, the use of single or combined homologous orheterologous promoter elements has been widely described in thescientific literature. The use of a regulatory element downstream of thetransgene was used for the sole purpose of putting a boundary whichmakes it possible to stop the process of transcription of the transgene,without presupposition as to their role as regards the quality or thequantity of the expression of the transgene.

[0003] The present invention relates to the use of an intron 1 isolatedfrom plant genes as a regulatory element, of now chimeric genescontaining them and to their use for the transformation of plants. Itrelates to an isolated DNA sequence capable of serving as a regulatoryelement in a chimeric gene which can be used for the transformation ofplants and allowing the expression of the product of translation of thechimeric gene in particular in the regions of the plant undergoing rapidgrowth, which comprises, in the direction of transcription of thechimeric gene, at least one intron such as the first intron (intron 1)of the noncoding 5′ region of a plant histone gene. It relates moreparticularly to the simultaneous use of the intron 1 as a regulatoryelement and of promoters isolated from the same plant gene. it allowsthe appropriate expression, both quantitative and qualitative, of thetransgenes under the control of these elements for gene regulation. Thisappropriate expression, obtained by the use of the present invention,may relate to characters such as; the resistance to pathogenic agentsfor crops, the resistance to phytotoxic plant-protection products, theproduction of substances of dietary or pharmacological interest. inparticular, it makes it possible to confer on the transgenic plants anenhanced tolerance to herbicides by a qualitative and quantitativepreferential expression of the product of expression of the chimericgenes in the regions of the plant undergoing rapid growth. This specificappropriate expression of the gene for herbicide resistance is obtainedby the simultaneous use of the promoter regulatory elements and of atleast one intron 1 of the histone gene of the “H3.3- like” type asregulatory element. Such a pattern of expression can be obtained for allthe characters which are of interest, as described above, with theregulatory elements used to confer an enhanced herbicide tolerance. Thepresent invention also relates to the plant cells transformed with theaid of these genes and the transformed plants regenerated from thesecells as well as the plants derived from crossings using thesetransformed plants.

[0004] Among the plant-protection products used for the protection ofcrops, the systemic products are characterized in that they aretransported in the plant after application and, for some of them,accumulate in the parts undergoing rapid growth, especially thecaulinary and root apices, causing, in the case of herbicides,deterioration, up to the destruction, of the sensitive plants. For someof the herbicides exhibiting this type of behaviour, the primary mode ofaction is known and results from inactivation of characterized enzymesinvolved in the biosynthesis pathways of compounds required for properdevelopment of the target plants. The target enzymes of those productsmay be located in various subcellular compartments and observation ofthe mode of action of known products most often shows a location in theplastid compartment.

[0005] Tolerance of plants sensitive to a product belonging to thisgroup of herbicides, and whose primary target is known, may be obtainedby stable introduction, into their genome, of a gene encoding the targetenzyme, of any phylogenetic origin, mutated or otherwise with respect tothe characteristics of inhibition, by the herbicide, of the product ofexpression of this gene. Another approach comprises introducing, in astable manner, into the genome of sensitive plants a gene of anyphylogenetic origin encoding an enzyme capable of metabolizing theherbicide into a compound which is inactive and nontoxic for thedevelopment of the plant. In the latter came, it is not necessary tohave characterized the target of the herbicide.

[0006] Given the mode of distribution and accumulation of products ofthis type in the treated plants, it is advantageous to be able toexpress the product of translation of these genes so as to allow theirpreferential expression and their accumulation in the regions of theplant undergoing rapid growth where these products accumulate.Furthermore, and in the case where the target of these products islocated in a cellular compartment other than the cytoplasm, it isadvantageous to be able to express the product of translation of thesegenes in the form of a precursor containing a polypeptide sequenceallowing directing of the protein conferring the tolerance into theappropriate compartment, and in particular in the plastid compartment.

[0007] By way of example illustrating this approach, there may bementioned glyphosate, sulfosate or fosametine which are broad-spectrumsystemic herbicides of the phosphonomethylglycine family. They actessentially as competitive inhibitors, in relation to PEP(phosphoenolpyruvate), of 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS, EC 2.5.1.19). After their application to the plant, they aretransported into the plant where they accumulate in the parts undergoingrapid growth, especially the caulinary and root apices, causing thedeterioration, up to the destruction, of the sensitive plants.

[0008] EPSPS, the principal target of these products, is an enzyme ofthe pathway of biosynthesis of aromatic amino acids which is located inthe plastid compartment. This enzyme is encoded by one or more nucleargenes and is synthesized in the form of a cytoplasmic precursor and thenimported into the plastids where it accumulates in its mature form.

[0009] The tolerance of plants to glyphosate and to products of thefamily is obtained by the stable introduction, into their genome, of anEPSPS gene of plant or bacterial origin, mutated or otherwise withrespect to the characteristics of inhibition, by glyphosate, of theproduct of this gene. Given the mode of action of glyphosate, it isadvantageous to be able to express the product of translation of thisgene so as to allow its high accumulation in the plastids and,furthermore, in the regions of the plant undergoing rapid growth wherethe products accumulate.

[0010] It is known, for example, from American patent 4,535,060 toconfer on a plant a tolerance to a herbicide of the above type, inparticular N-phosphonomethylglycine or glyphosate, by introduction, intothe genome of the plants, of a gene encoding an EPSPS carrying at leastone mutation making this enzyme more resistant to its competitiveinhibitor (glyphosate), after location of the enzyme in the plastidcompartment. These techniques require, however, to be improved forgreater reliability in the use of these plants during a treatment withthese products under agronomic conditions.

[0011] In the present description, “plant” is understood to mean anydifferentiated multicellular organism capable of photosynthesis and“plant cell” any cell derived from a plant and capable of constitutingundifferentiated tissues such as calli, or differentiated tissues suchas embryos or plant portions or plants or seeds. “Intron 1 ofArabidopsis as a regulatory element” is understood to mean an isolatedDNA sequence of variable length, situated upstream of the coding part orcorresponding to the structural part of a transcribed gene. Gene fortolerance to a herbicide is understood to mean any gene, of anyphylogenetic origin, encoding either the target enzyme for theherbicide, optionally having one or more mutations with respect to thecharacteristics of inhibition by the herbicide, or an enzyme capable ofmetabolizing the herbicide into a compound which is inactive andnontoxic for the plant. Zones of the plants undergoing rapid growth areunderstood to mean the regions which are the seat of substantial cellmultiplications, in particular the apical regions.

[0012] The present invention relates to the production of transformedplants having an enhanced tolerance to herbicides accumulating in thezones of the treated plants undergoing rapid growth, by regeneration ofcells transformed with the aid of new chimeric genes comprising a genefor tolerance to these products. The subject of the invention is alsothe production of transformed plants having an enhanced tolerance toherbicides of the phosphonomethylglycine family by regeneration of cellstransformed with the aid of new chimeric genes comprising a gene fortolerance to these herbicides. The invention also relates to these newchimeric genes, as well as to transformed plants which are more tolerantbecause of a better tolerance in the parts of these plants undergoingrapid growth, as well as to the plants derived from crossings usingthese transformed plants. Its subject is also new intron 1 of a planthistone and its use as regulatory zone for the construction of the abovechimeric genes.

[0013] More particularly, the subject of the invention is a chimericgene for conferring on plants especially an enhanced tolerance to aherbicide having EPSPS as target, comprising, in the direction oftranscription, a promoter element, a signal peptide sequence, a sequenceencoding an enzyme for tolerance to the products of thephosphonomethylglycine family and a regulatory element, characterized inthat the regulatory element comprises a fragment of an intron 1 of aplant histone gene in any orientation relative to its initialorientation in the gene from which it is derived, allowing thepreferential expression and the accumulation of the protein fortolerance to the herbicide in the zones for accumulation of the saidherbicide.

[0014] The histone gene, from which intron 1 according to the inventionis derived, comes from a monocotyledonous plant such an for examplewheat, maize or rice, or preferably from a dicotyledonous plant such asfor example lucerne, sunflower, soya bean, rapeseed or preferablyArabidopsis thaliana. Preferably, a histone gene of the “H3.3-like” typeis used.

[0015] The signal peptide sequence comprises, in the direction oftranscription, at least one signal peptide sequence of a plant geneencoding a signal peptide directing transport of a polypeptide to aplastid, a portion of the sequence of the mature N-terminal part of aplant gene produced when the first signal peptide is cleaved byproteolytic enzymes, and then a second signal peptide of a plant geneencoding a signal peptide directing transport of the polypeptide to asub-compartment of the plastid. The signal peptide sequence ispreferably derived from a gene for the small subunit ofribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisco) according toEuropean patent application PCT 508 909. The role of this characteristicsequence is to allow the release, into the plastid compartment, of amature polypeptide with a maximum efficiency, preferably in a nativeform.

[0016] The coding sequence which can be used in the chimeric geneaccording to the invention comes from a herbicide tolerance gene of anyphylogenetic origin. This sequence may be especially that of the mutatedEPSPS having a degree of tolerance to glyphosate.

[0017] The promoter element according to European patent application PCT507 698 may be of any origin, in a single or duplicated or combined formof a gene naturally expressed in plants, that is to say, for example ofbacterial origin such as that of the nopaline synthase gene, or of viralorigin such as that of the 35S transcript of the cauliflower mosaicvirus, or preferably of plant origin such as that of the small subunitof the ribulose-1,5-bisphosphate carboxylase/oxygenase or preferablysuch as that of a plant histone gene and preferably from Arabidopsisthaliana. A histone gene of the “H4” type is preferably used.

[0018] The chimeric gene according to the invention may comprise, inaddition to the above essential parts, an untranslated intermediate zone(linker) between the promoter zone and the coding zone as well asbetween the coding zone and intron 1 and which may be of anyphylogenetic origin.

[0019] The following examples show by way of illustration, but with nolimitation being implied, several aspects of the invention: isolation ofthe introns according to the invention and their use for the genetictransformation of plants as well as the improved qualities of expressionof the heterologous genes of plants transformed with the aid of theseintrons. References to “Current Protocols in Molecular Biology” are toVolumes 1 and 2, Ausubel F. M. et al., published by Greene Publishingassociates and Wiley Interscience (1989) (CPMB).

EXAMPLE 1

[0020] 1. Production of an EPSPS fragment from Arabidopsis thaliana

[0021] a) two 20-mer oligonucleotides of respective sequences:5′-GCTCTGCTCATGTCTGCTCC-3′ 5′-GCCCGCCCTTGACAAAGAAA-3′

[0022] were synthesized from the sequence of an EPSPS gene fromArabidopsis thaliana (Klee H. J. et al., (1987) Mol. Gen. Genet., 210,437-442). These two oligonucleotides correspond to positions 1523 to1543 and 1737 to 1717, respectively, of the published sequence and inconvergent orientation.

[0023] b) The total DNA from Arabidopsis thaliana (var. columbia) wasobtained from Clontech (catalogue reference: 6970-1)

[0024] c) 50 nanograms (ng) of DNA are mixed with 300 ng of each of theoligonucleotides and subjected to 35 amplification cycles with aPerkin-Elmer 9400 apparatus under the standard medium conditions foramplification recommended by the supplier. The resulting 204 bp fragmentconstitutes the EPSPS fragment from Arabidopsis thaliana.

[0025] 2. Construction of a library of a cDNA from a BMS maize cellline.

[0026] a) 5 g of filtered cells are ground in liquid nitrogen and thetotal nucleic acids extracted according to the method described by Shureet al. with the following modifications:

[0027] the pH of the lysis buffer is adjusted to pH=9.0

[0028] after precipitation with isopropanol, the pellet is taken up inwater and after dissolution, adjusted to 2.5 M LiCl. After incubationfor 12 h at 0° C., the pellet from the 15 min centrifugation at 30,000 gat 4° C. is resolubilized. The LiCl precipitation stage is thenrepeated. The resolubilized pellet constitutes the RNA fraction of thetotal nucleic acids.

[0029] b) the RNA-poly A+ fraction of the RNA fraction is obtained bychromatography on an oligo-dT cellulose column as described in “CurrentProtocols in Molecular Biology”.

[0030] c) Synthesis of double-stranded cDNA with an EcoRI synthetic end;it is carried out by following the procedure of the supplier of thevarious reagents necessary for this synthesis in the form of a kit: the“copy kit” from the company Invitrogen.

[0031] Two single-stranded and partially complementary oligonucleotidesof respective sequences: 5′-AATTCCCGGG-3′ 5′-CCCGGG-3′

[0032] (the latter being phosphorylated) are ligated to double-strandedcDNAs with blunt ends.

[0033] This ligation of the adaptors results in the creation of SmaIsites attached to the double-stranded cDNAs and of EcoRI sites incohesive form at each and of the double-stranded cDNAs.

[0034] d) Creation of the library:

[0035] The cDNAs having at their ends the cohesive artificial EcoRIsites are ligated to the λgt10 bacteriophage cDNA cut with EcoRI anddephosphorylated according to the procedure of the supplier New EnglandBiolabs.

[0036] An aliquot from the ligation reaction was encapsidated in vitrowith encapsidation extracts: Gigapack Gold according to the supplier'sinstructions, this library was titrated using the bactertium E. coliC600 hfl. The library thus obtained is amplified and stored according tothe instructions of the same supplier and constitutes the cDNA libraryfrom BMS maize cell suspension.

[0037] 3. screening of the cDNA library from BMS maize cell suspensionwith the EPSPS probe from Arabidopsis thaliana:

[0038] The procedure followed is that of “Current Protocols in MolecularBiology”. Briefly, about 10⁶ recombinant phages are plated on an LBplate at a mean density of 100 phages/cm². The lysis plaques arereplicated in duplicate on a Hybond N membrane from Amersham.

[0039] The DNA was fixed onto the filters by a 1600 kJ UV treatment(Stratalinker from Stratagene). The filters were prehybridized in:6×SSC/0.1% SDS/0.25[lacuna] skimmed milk for 2 h at 65° C. The EPSPSprobe from Arabidopsis thaliana was labelled with ³²P-dCTP by randompriming according to the instructions of the supplier (Kit Ready to Gofrom Pharmacia). The specific activity obtained is of the order of 10³cpm per μg of fragment. After denaturation for 5 min at 100° C., theprobe is added to the prehybridization medium and the hybridization iscontinued for 14 hours at 55° C. The filters are fluorographed for 48 hat −80° C. with a Kodak XAR5 film and intensifying screens HyperscreenRPN from Amersham. The alignment of the positive spots on the filterwith the plates from which they are derived make it possible to collect,from the plate, the zones corresponding to the phages exhibiting apositive hybridization response with the EPSPS probe from Arabidopsisthaliana. This step of plating, transfer, hybridization and recovery isrepeated until all the spots of the plate of phages successivelypurified prove 100% positive in hybridization. A lysis plaque perindependent phage in then collected in the diluent λ medium (Tris-ClpH=7.5; 10 mM MgSO4; 0.1 M NaCl; 0.1% gelatine), these phages insolution constituting the positive EPSPS clones from the BMS maize cellsuspension.

[0040] 4. Preparation and analysis of the DNA of the EPSPS clones fromthe BMS maize cell suspension.

[0041] About 5×10³ phages are added to 20 ml of 600hfl bacteria at OD 2(600 nm/ml) and incubated for 15 minutes at 37° C. This suspension isthen diluted in 200 ml of growth medium for the bacteria in a 1lErlenmeyer flask and shaken in a rotary shaker at 250 rpm. Lysis isobserved by clarification of the medium, corresponding to lysis of theturbid bacteria and occurs after about 4 h of shaking. This supernatantis then treated as described in “Current Protocols in MolecularBiology”. The DNA obtained corresponds to the EPSPS clones from the BMSmaize cell suspension.

[0042] One to two μg of this DNA are cut with EcoRI and separated on a0.8% LGTA/TBE agarose gel (ref. CPMB). A final verification consists inensuring that the purified DNA indeed exhibits a hybridization signalwith the EPSPS probe from Arabidopsis thaliana. After electrophoresis,the DNA fragments are transferred onto Hybond N membrane from Amershamaccording to the Southern procedure described in “Current Protocols inMolecular Biology”. The filter is hybridized with the EPSPS probe fromArabidopsis thaliana according to the conditions described in paragraph3 above. The clone exhibiting a hybridization signal with the EPSPSprobe from Arabidopsis thaliana and containing the longest EcoRIfragment has a gel-estimated size of about 1.7 kbp.

[0043] 5. Production of the pRPA-ML-711clone:

[0044] Ten μg of DNA from the phage clone containing the 1.7 kbp insertare digested with EcoRI and separated on a 0.8% LGTA/TBE agarose gel(ref. CPMB). The gel fragment containing the 1.7 kbp insert is excisedfrom the gel by BET staining and the fragment is treated with β-agaraseaccording to the procedure of the supplier New England Biolabs. The DNApurified from the 1.7 kbp fragment is ligated at 12° C. for 14 h withDNA from the plasmid pUC 19 (New England Biolabs) cut with EcoRIaccording to the ligation procedure described in “Current Protocols inMolecular Biology”. Two μl of the above ligation mixture are used forthe transformation of one aliquot of electrocompetent E. coli DH10B; thetransformation occurs by electroporation using the following conditions:the mixture of competent bacteria and ligation medium is introduced intoan electroporation cuvette 0.2 cm thick (Biorad) previously cooled to 0°C. The physical electroporation conditions using an electroporator ofBiorad trade mark are 2500 volts, 25 μFarad and 200 Ω. Under theseconditions, the mean condenser discharge time is of the order of 4.2milliseconds. The bacteria are then taken up in 1 ml of SOC medium (ref.CPMB) and shaken for 1 hour at 200 rpm on a rotary shaker in 15 mlCorning tubes. After plating on LB/agar medium supplemented with 100μg/ml of carbenicillin, the mini-preparations of the bacteria cloneshaving grown overnight at 37° C. are carried out according to theprocedure described in “Current Protocols in Molecular Biology”. Afterdigestion of the DNA with EcoRI and separation by electrophoresis on a0.8% LGTA/TBE agarose gel (ref. CPMB), the clones having a 1.7 kbpinsert are conserved. A final verification consists in ensuring that thepurified DNA indeed exhibits a hybridization signal with the EPSPS probefrom Arabidopsis thaliana. After electrophoresis, the DNA fragments aretransferred onto a Hybond N membrane from Amersham according to theSouthern procedure described in “Current Protocols in MolecularBiology”. The filter is hybridized with the EPSPS probe from Arabidopsisthaliana according to the conditions described in paragraph 3 above. Theplasmid alone having a 1.7 kbp insert and hybridizing with the EPSPSprobe from Arabidopsis thaliana was prepared on a larger scale and theDNA resulting from the lysis of the bacteria purified on a CsCl gradientas described in “Current Protocols in Molecular Biology”. The purifiedDNA was partially sequenced with a Pharmacia kit, following thesupplier's instructions and using, as primers, the direct and reverseM13 universal primers ordered from the same supplier. The partialsequence produced covers about 0.5 kbp. The derived amino acid sequencein the region of the mature protein (about 50 amino acid residues)exhibits 100% identity with the corresponding amino sequence of themature maize EPSPS described in American patent U.S. Pat. No. 4,971,908.This clone, corresponding to a 1.7 kbp EcoRI fragment of the DNA for theEPSP from the BMS maize cell suspension, was called pRPA-ML-711. Thecomplete sequence of this clone was obtained on both strands by usingthe Pharmacia kit procedure and by synthesizing oligonucleotides whichare complementary and of opposite direction every 250 bp approximately.The complete sequence of this 1713 bp clone obtained is presented by SEQID No. 1.

[0045] 6. Production of the clone pRPA-ML-715:

[0046] Analysis of the sequence of the clone pRPA-ML-711 and inparticular comparison of the derived amino acid sequence with that frommaize shows a sequence extension of 92 bp upstream of the GCG codonencoding the NH₂-terminal alanine of the mature part of the maize EPSPS(American patent U.S. Pat. No. 4,971,908). Likewise, a 288 bp extensiondownstream of the AAT codon encoding the COOH-terminal asparagine of themature part of the maize EPSPS (American patent U.S. Pat. No. 4,971,908)is observed. These two parts might correspond, for the NH₂-terminalextension, to a portion of the sequence of a signal peptide beforeplastid location and, for the COOH-terminal extension, to theuntranslated 3′ region of the cDNA.

[0047] In order to obtain a cDNA encoding the mature part of the cDNAfor the maize EPSPS, as described in U.S. Pat. No. 4,971,908, thefollowing operations were carried out:

[0048] a) Elimination of the untranslated 3′ region: construction ofpRPA-ML-712:

[0049] The clone pRPA-ML-711 was cut with the restriction enzyme AseIand the resulting ends of this cut made blunt by treating with theKlenow fragment of DNA polymerase I according to the procedure describedin CPMB. A cut with the restriction enzyme SacII was then performed. TheDNA resulting from these operations was separated by electrophoresis ona 1% LGTA/TBE agarose gel (ref. CPMD)

[0050] The gel fragment containing the insert “AseI-blunt ends/SacII” of0.4 kbp was excised from the gel and purified according to the proceduredescribed in paragraph 5 above. The DNA of the clone pRPA-ML-711 was cutwith the restriction enzyme HindIII situated in the polylinker of thecloning vector pUC19 and the ends resulting from this cut were madeblunt by treating with the Klenow fragment of DNA polymerase I. A cutwith the restriction enzyme SacII was then performed. The DNA resultingfrom these manipulations was separated by electrophoresis on a 0.7%LGTA/TBE agarose gel (ref. CPMB).

[0051] The gel fragment containing the insert HindIII-blunt ends/SacIIof about 3.7 kbp was excised from the gel and purified according to theprocedure described in paragraph 5 above.

[0052] The two inserts were ligated, and 2 μl of the ligation mixtureserved to transform E. coli DH10B as described above in paragraph 5.

[0053] The plasmid DNA content of the various clones was analysedaccording to the procedure described for pRPA-ML-711. One of the plasmidclones retained contains an EcoRI-HindIII insert of about 1.45 kbp. Thesequence of the terminal ends of this clones shows that the 5′ end ofthe insert corresponds exactly to the corresponding end of pRPA-ML-711and that the 3′ terminal end has the following sequence:

[0054] “5′. . . AATTAAGCTCTAGAGTCGACCTGCAGGCATGCAAGCTT-3′”.

[0055] The sequence underlined corresponds to the codon for theCOOH-terminal amino acid asparagine, the next codon corresponding to thestop codon for translation. The nucleotides downstream correspond tosequence components of the polylinker of pUC19. This clone, comprisingthe sequence of pRPAML-711 up to the site for termination of translationof the mature maize EPSPS and followed by sequences of the polylinker ofpUC19 up to the HindIII site, was called pRPA-ML-712.

[0056] b) Modification of the 5′ end of pRPA-ML-712: construction ofpRPA-ML-715

[0057] The clone pRPA-NL-712 was cut with the restriction enzymes PstIand HindIII. The DNA resulting from these manipulations was separated byelectrophoresis on a 0.8% LGTA/TBE agarose gel (ref. CPMB). The gelfragment containing the PstI/EcoRI insert of 1.3 kbp was excised fromthe gel and purified according to the procedure described in paragraph 5above. This insert was ligated in the presence of an equimolar quantityof each of the two partially complementary oligonucleotides of sequence:Oligo 1: 5′-GAGCCGAGCTCCATGGCCGGCGCCGAGGAGATCGTGCTGCA-3′ Oligo 2:5′-GCACGATCTCCTCGGCGCCGGCCATGGAGCTCGGCTC-3′

[0058] as well as in the presence of DNA from the plasmid pUC19 digestedwith the restriction enzymes BamHI and HindIII.

[0059] Two μl of the ligation mixture served to transform E. coli DH10Bas described above in paragraph 5. After analysis of the plasmid DNAcontent of various clones according to the procedure described above inparagraph 5, one of the clones having an insert of about 1.3 kbp wasconserved for subsequent analyses. The sequence of the terminal 5′ endof the clone retained shows that the DNA sequence in this region is thefollowing: sequence of the polylinker of pUC19 of the EcoRI to BamHIsites, followed by the sequence of the oligonucleotides used during thecloning, followed by the rest of the sequence present in pRPAML-712.This clone was called pRPA-ML-713. This clone has a methionine codon ATGincluded in an NcoI site upstream of the N-terminal alanine codon of themature EPSPSynthase. Furthermore, the alanine and glycine codons of theN-terminal and were conserved, but modified on the third variable base:initial GCGGGT gives modified GCCGGC.

[0060] The clone pRPA-ML-713 was cut with the restriction enzyme HindIIIand the ends of this cut made blunt by treating with the Klenow fragmentof DNA polymerase I. A cut with the restriction enzyme SacI was thenperformed. The DNA resulting from these manipulations was separated byelectrophoresis on a 0.8% LGTA/TBE agarose gel (ref. CPMB) The gelfragment containing the insert “HindIII-blunt ends/SacI” of 1.3 kbp wasexcised from the gel and purified according to the procedure describedin paragraph 5 above. This insert was ligated in the presence of DNAfrom the plasmid pUC19 digested with the restriction enzyme XbaI and theends of this cut made blunt by treating with the Klenow fragment of DNApolymerase I. A cut with the restriction enzyme SacI was then performed.Two μl of the ligation mixture served to transform E. coli DH10B asdescribed above in paragraph 5. After analysis of the plasmid DNAcontent of various clones according to the procedure described above inparagraph 5, one of the clones having an insert of about 1.3 kbp wasconserved for subsequent analyses. The sequence of the terminal ends ofthe clone retained shows that the DNA sequence is the following:sequence of the polylinker of pUC19 of the EcoRI to SacI sites, followedby the sequence of the oligonucleotides used during the cloning, fromwhich the 4 bp GATCC of oligonucleotide 1 described above have beendeleted, followed by the rest of the sequence present in pRPA-ML-712 upto the HindIII site and sequence of the polylinker of pUC19 from XbaI toHindIII. This clone was called pRPA-NL-715.

[0061] 7) Production of a cDNA encoding a mature

[0062] All the mutagenesis steps were carried out with the U.S.E.mutagenesis kit from Pharmacia, following the instructions of thesupplier. The principle of this mutagenesis system is as follows: theplasmid DNA is heat-denatured and recombined in the presence of a molarexcess, on the one hand, of the mutagenesis oligonucleotide and, on theother hand, of an oligonuclootide which makes it possible to eliminate aunique restriction enzyme site present in the polylinker. After thereassociation step, the synthesis of the complementary strand isperformed by the action of T4 DNA polymerase in the presence of T4 DNAligase and protein of gene 32 in an appropriate buffer provided. Thesynthesis product is incubated in the presence of the restrictionenzyme, whose site is supposed to have disappeared by mutagenesis. TheE. coli strain exhibiting, in particular, the mutS mutation is used ashost for the transformation of this DNA. After growth in liquid medium,the total plasmid DNA is prepared and incubated in the presence of therestriction enzyme used above. After these treatments, the E. coli DH10Bstrain is used as host for the transformation. The plasmid DNA of theisolated clones is prepared and the presence of the mutation introducedis checked by sequencing.

[0063] A) Site or sequence modifications with no effect a priori on theresistance character of maize EPSPS to the products which arecompetitive inhibitors of the activity of EPSP synthase: elimination ofan internal NcoI site from pRPA-ML-715.

[0064] The sequence of pRPA-ML-715 is arbitrarily numbered by placingthe first base of the N-terminal alanine codon GCC in position 1. Thissequence has an NcoI site in position 1217. The site-modifyingoligonucleotide has the sequence;

[0065] 5′-CCACAGGATGGCGATGGCCTTCTCC-3′.

[0066] After sequencing according to the references given above, thesequence read after mutagenesis corresponds to that of theoligonucleotide used. The NcoI site was indeed eliminated andtranslation into amino acids in this region conserves the initialsequence present in pRPA-ML-715.

[0067] This clone was called pRPA-ML-716.

[0068] The 1340 bp sequence of this clone is represented as SEQ ID No. 2and SEQ ID No. 3.

[0069] B) Sequence modifications allowing an increase in the resistancecharacter of maize EPSPS to products which are competitive inhibitors ofthe activity of EPSP synthase.

[0070] The following oligonucleotides were used: a) Thr 102 → Ilemutation. 5′-GAATGCTGGAATCGCAATGCGGCCATTGACAGC-3′ b) Pro 106 → Sermutation. 5′-GAATGCTGGAACTGCAATGCGGTCCTTGACAGC-3′ c) Gly 101 → Ala andThr 102 → Ile mutations. 5′-CTTGGGGAATGCTGCCATCGCAATGCGGCCATTG-3′ d) Thr102 → Ile and Pro 106 → Ser mutations.5′-GGGGAATGCTGGAATCGCAATGCGGTCCTTGACAGC-3′

[0071] After sequencing, the sequence read after mutagenesis on thethree mutated fragments is identical to the sequence of the parental DNApRPA-ML-716 with the exception of the mutagenesis region whichcorresponds to that of the mutagenesis oligonucleotides used. Theseclones were called: pRPA-ML-717 for the Thr 102→Ile mutation,pRPA-ML-718 for the Pro 106→Ser mutation, pRPA-ML-719 for the Gly101→Ala and Thr 102→Ile mutations and pRPA-ML-720 for the Thr 102→Ileand Pro 106→Ser mutations.

[0072] The 1340 bp sequence of pRPA-ML-720 is represented as SEQ ID No.4 and SEQ ID No. 5.

[0073] The NcoI-HindIII insert of 1395 bp will be called in the rest ofthe descriptions “the double mutant of maize EPSPS”.

EXAMPLE 2 Construction of chimeric genes

[0074] The construction of chimeric genes according to the invention iscarried out using the following elements:

[0075] 1). The genomic clone (cosmid clone c22) from Arabidopsisthaliana, containing two genes of the “H3.3-like” type was isolated asdescribed in Chaubet et al. (J. Mol. Biol. 1992, 225 569-574).

[0076] 2). Intron No. 1:

[0077] A DNA fragment of 414 base pairs is purified from digestion ofthe cosmid clone c22 with the restriction enzyme DdeI followed bytreatment with a Klenow fragment of DNA polymerase from E. coli,according to the manufacturer's instructions for creating a blunt-endedDNA fragment and then cut with MseI. The purified DNA fragment isligated to a synthetic oligonucleotide adapter having the followingsequence: Adaptor 1: 5′ TAATTTGTTGAACAGATCCC 3′      TAAACAACTTGTCTAGGG

[0078] The ligation product is cloned into pGEM7Zf (+) (Stratagenecatalogue No. P2251) which was digested with SmaI. This clone, called“intron No. 1”, is checked by sequencing (SEQ ID No. 6).

[0079] 3). Intron No. 2:

[0080] A DNA fragment of 494 base pairs is purified from the digestionof the cosmid clone c22 with the restriction enzymes AluI and CfoI. Thepurified DNA fragment is listed to a synthetic oligonucleotide adaptorhaving the following sequence: Adaptor 2: 5′   CAGATCCCGGGATCTGCG 3′   GCGTCTAGGGCCCTAGACGC

[0081] The ligation product is cloned into pGEM7Zf (+) (Stratagenecatalogue No. P2251) which was digested with SmaI. This clone, called“intron No. 2”, is checked by sequencing (SEQ ID No. 7).

[0082] 4). pRA-1

[0083] The construction of this plasmid is described in French patent9,308,029. This plasmid is a derivative of pBI 101.1 (Clonetechcatalogue No. 6017-1) which contains the histone promoter fromArabidopsis H4A748 regulating the synthesis of the E. coliβ-glucoronidase gene and of the nopaline synthase (“NOS”)polyadenylation site. Thus, a chimeric gene is obtained having thestructure:

[0084] “H4A748 promoter-GUS gene-NOS”

[0085] 5). pCG-1

[0086] This plasmid contains the above intron No. 1 placed between theH4A748 promoter and the GUS coding region of pRA-1. This plamid isobtained by digestion of cosmid clone c22 with BamHI and SmaI. Theintron No. 1 of 418 base pairs is directly ligated into pRA-1 which wasdigested with BamHI and SmaI.

[0087] Thus, a chimeric gene in obtained having the structure:

[0088] “H4A748 promoter-intron NO. 1-GUS gene-NOS”

[0089] 6). pCG-13

[0090] This plasmid contains the above intron No. 2 placed between theH4A748 promoter and the GUS coding region of pRA-1. This plasmid isobtained by digestion of cosmid clone c22 with BamHI and SmaI. Theintron No. 2 of 494 base pairs is directly ligated into pRA-1 which wasdigested with BamHI and SmaI.

[0091] Thus, a chimeric gene is obtained having the structure:

[0092] “H4A748 promoter-intron No. 2-GUS gene-NOS”

[0093] 7). pCG-15

[0094] This plasmid contains only intron No. 1 before the above GUScoding sequence placed between the H4A748 promoter and the GUS codingregion of pCG-1. This plasmid is obtained by digestion of pCG-1 withBamHI and HindIII followed by treatment with a Klenow fragment of DNApolymerase from E. coli, according to the manufacturer's instructionsfor creating a blunt-ended DNA fragment.

[0095] This vector is then religated to give a chimeric gene having thestructure:

[0096] “intron No. 1-GUS-NOS”

[0097] 8). pCG-18

[0098] This plasmid contains only the above intron No. 2 in front of theGUS coding sequence of pCG-13. This plasmid is obtained by partialdigestion of pCG-13 with BamHI and SphI, followed by treatment with afragment of T4 phage DNA polymerase, according to the manufacturer'sinstructions in order to create a blunt-ended DNA fragment.

[0099] This vector is then religated and checked by enzymatic digestionin order to give a chimeric gene having the structure:

[0100] “intron No. 2-GUS-NOS”

[0101] 9). pRPA-RD-124

[0102] Addition of a “nos” polyadenylation signal to pRPA-ML-720 withcreation of a cloning cassette containing the maize double mutant EPSPSgene (Thr 102→Ile and Pro 106→ser). pRPA-ML-720 is digested with HindIIIand treated with the Klenow fragment of DNA polymerase from E. coli inorder to produce a blunt end. A second digestion is carried out withNcoI and the EPSPS fragment is purified. The EPSPS gene is then ligatedwith purified pRPA-RD-12 (a cloning cassette containing the nopalinesynthase polyadenylation signal) to give pRPA-RD-124. To obtain thepurifed useful vector pRPA-RD-12, it was necessary for the latter to bepreviously digested with SalI, treated with Klenow DNA polymerase, andthen digested a second time with NcoI.

[0103] 10). pRPA-RD-125

[0104] Addition of an optimized signal peptide (OSP) from pRPA-RD-124with creation of a cloning cassette containing the EPSPS gene targetedon the plasmids. pRPA-RD-7 (European Patent Application EP 652 286) isdigested with SphI, treated with T4 DNA polymerase and then digestedwith SpeI and the OSP fragment is purified. This OSP fragment is clonedinto pRPA-RD-124 which was previously digested with NcoI, treated withKlenow DNA polymerase in order to remove the 3′ protruding part, andthen digested with SpeI. This clone is then sequenced in order to ensurethe correct translational fusion between the OSP and the EPSPS gene.pRPA-RD-125 is then obtained.

[0105] 11). pRPA-RD-196

[0106] In this plasmid, the “intron No. 1+β-glucoronidase gene from E.coli” portion of pCG-1 is replaced by a chimeric gene of 2 kilobasescontaining an optimized signal peptide, a double mutant EPSPS gene(Ile₁₀₂+Ser₁₀₆) and a nopaline synthase polyadenylation site (“NOS”)isolated from pRPA-RD-125. To obtain pRPA-RD-196, the digestion of pCG-1is performed with EcoRI and BamHI, followed by treatment with a Klenowfragment of DNA polymerase from E. coli, according to the manufacturer'sinstructions in order to create a blunt-ended DNA fragment. The2-kilobase DNA fragment containing an optimized signal peptide of adouble mutant EPSPS gene (Ile₁₀₂+Ser₁₀₆) and a nopaline synthasepolyadenylation site (“NOS”) is obtained from pRPA-RD-125 by digestionwith NcoI and NotI, followed by treatment with DNA polymerase from E.coli, according to the manufacturer's instructions in order to create ablunt-ended DNA fragment. This blunt-ended fragment is then ligated intopCG-1 prepared above.

[0107] A chimeric gene is thus obtained having the structure:

[0108] “H4A748 promoter-OSP-maize EPSPS gene-NOS”

[0109] 12). pRPA-RD-197

[0110] In this plasmid, the “β-glucoronidase gene from E. coli portionof pCG-1 is replaced by a chimeric gene of 2 kilobases containing anoptimized signal peptide, a double mutant EPSPS gene (Ile₁₀₂+Ser₁₀₆) anda nopaline synthase polyadenylation site (“NOS”) isolated frompRPA-RD-125. To obtain pRPA-RD-197, the digestion of pCG-1 is performedwith EcoRI, followed by treatment with a Klenow fragment of DNApolymerase from E. coli, according to the manufacturer's instructions inorder to create a blunt-ended DNA fragment, then cut with SmaI. The2-kilobase DNA fragment containing an optimized signal peptide, a doublemutant EPSPS gene (Ile₁₀₂+Ser₁₀₆”) and a nopaline synthasepolyadenylation site (“NOS”) is obtained from pRPA-RD-125 by digestionwith NcoI and NotI, followed by a treatment with DNA polymerase from E.coli, according to the manufacturer's instructions in order to create ablunt-ended DNA fragment. This blunt-ended fragment is then ligated intopCG-1 prepared above.

[0111] A chimeric gene is thus obtained having the structure:

[0112] “H4A74B promoter-intron No. 1-maize EPSPS gene-NOS”

[0113] 13). pRPA-RD-198

[0114] In this plasmid, the “β-glucoronidase gene from E. coli” portionof pCG-13 is replaced by a chimeric gene of 2 kilobases containing anoptimized signal peptide, a double mutant EPSPS gene (Ile₁₀₂+Ser₁₀₆) anda nopaline synthase polyadenylation site (“NOS”) isolated frompRPA-RD-125. To obtain pRPA-RD-198, the digestion of pCG-13 is performedwith EcoRI, followed by treatment with a Klenow fragment of DNApolymerase from E. coli, according to the manufacturer's instructions inorder to create a blunt-ended DNA fragment, then cut with SmaI. The2-kilobase DNA fragment containing an optimized signal peptide, a doublemutant EPSPS gene (Ile₁₀₂+Ser₁₀₆) and a nopaline synthasepolyadenylation site (“NOS”) is obtained from pRPA-RD-123 by digestionwith NcoI and NotI, followed by a treatment with DNA polymerase from E.coli, according to the manufacturer's instructions in order to create ablunt-ended DNA fragment. This blunt-ended fragment is then ligated intopCG-13 prepared above.

[0115] A chimeric gene is thus obtained having the structure:

[0116] H4A74B promoter-intron No. 2-OSP-maize EPSPS gene-“NOS”

EXAMPLE 3 Expression of the activity of a reporter gene

[0117] 1) Transformation and regeneration

[0118] The vector is introduced into the nononcogenic strain ofAgrobacterium tumefaciens LBA 4404 available from a catalogue (Clontech#6027-1) by triparental crossing using the “helper” plasmid pRK 2013 inEscherichia coil HB101 according to the procedure described by Bevan M.(1984) Nucl. Acids Res., 12, 8711-8721.

[0119] The transformation technique using root explants of Arabidopsisthaliana L.-ecotype C24 was carried out according to the proceduredescribed by Valvekens D. et al. (1988) Proc. Natl. Acad. Sci USA, 85,5536-5540. Briefly, 3 steps are necessary: induction of the formation ofcalli on Gamborg B5 medium supplemented with 2,4-D and kinetin;formation of buds on Gamborg B5 medium supplemented with 2iP and IAA;rooting and formation of seeds on hormone-free M5.

[0120] 2) Measurement of the GUS activity in plants

[0121] a—histochemical observations

[0122] Visualization of the GUS activity by histochemical spots(Jefferson R. A. et al. (1987) EMBO J. 6, 3901-3907) on 10-daytransgenic plants shows an increase in the intensity of thehistochemical pattern which is tissue-specific for the plasmidscontaining the intron sequences (pCG-1 and pCG-13) compared with thosewithout these introns (pRA-1). In particular, the pattern of spots forpCG-1 and pCG-13 is identical, showing an increase in intensity of thespots for the vascular and meristematic tissues, leaves and rootscompared with that of the construct pRA-1. The constructs containingonly the sequences of intron No. 1 (pCG-15 and pCG-18) show an extremelyclear histochemical spot only in the apical meristem region.

[0123] b—fluoromeatric measurements

[0124] The GUS activity measured by fluorometry on extracts of floraland leaf buds of the rosette (Jefferson R. A. et al. (1987) EMBO J., 6,3901-3907) from 12 plants, shows that the activity of the H4A74Bpromoter is increased under the influence of intron Nos. 1 and2.Compared with the construct pRA-1, the GUS activity of pCG-1 andpCG-13 are at least six times greater in the floral buds, twenty timesgreater in the leaves of the rosette and twenty-six times greater in theroots.

[0125] These measurements clearly show that introns Nos. 1 and 2 ofArabidopsis histone genes of the “H3.3-like” type used as a regulatoryelement induces an increase in the activity of expression of thechimeric gene.

EXAMPLE 4 Tolerance of transgenic plants to a herbicide

[0126] 1) Transformation and regeneration

[0127] The vector is introduced into the nononcogenic strain ofAgrobacterium tumefaciens LBA 4404 available from a catalogue (Clontech#6027-1) by triparental crossing using the “helper” plasmid pRK 2013 inEscherichia coli HB101 according to the procedure described by Bevan M.(1984) Nucl. Acids Res., 12, 8711-8721.

[0128] The transformation technique using foliar explants of tobacco isbased on the procedure described by Horsh R. et al. (1985) Science, 227,1229-1231. The regeneration of the PBD6 tobacco (origin SEITA-France)from foliar explants is carried out on a Murashige and Skoog (MS) basalmedium comprising 30 g/l of sucrose as well as 200 μg/ml of kanamycin inthree successive steps: the first comprises the induction of shoots onan MS medium supplemented with 30 g of sucrose containing 0.05 mg ofnaphthylacetic acid (NAA) and 2 mg/l of benzylaminopurine (BAP) for 15days. The shoots formed during this step are then developed by culturingon an MS medium supplemented with 30 g/l of sucrose but not containingany hormone, for 10 days. The developed shoots are then removed and theyare cultured on an MS rooting medium diluted one half, with half thecontent of salts, vitamins and sugars and not containing any hormone.After about 15 days, the rooted shoots are planted in the soil.

[0129] 2) Measurement of the tolerance to glyphosate:

[0130] Twenty transformed plants were regenerated and transferred to agreenhouse for each of the constructs pRPA-RD-196, pRPA-RD-197 andpRPA-RD-198. These plants were treated in a greenhouse at the 5-leafstage with an aqueous suspension of herbicide, sold under the trademarkRoundUp, corresponding to 0.8 kg of active substance glyphosate perhectare.

[0131] The results correspond to the observation of phytotoxicity valuesnoted 3 weeks after treatment. Under these conditions, it is observedthat the plants transformed with the constructs have on average anacceptable tolerance (pRPA-RD-196) or even a good tolerance (pRPA-RD-197and pRPA-RD-196) whereas the untransformed control plants are completelydestroyed.

[0132] These results show clearly the improvement offered by the use ofa chimeric gene according to the invention for the same gene encodingtolerance to glyphlosate.

[0133] The transformed plants according to the invention may be used asparents for producing lines and hybrids having the phenotypic charactercorresponding to the expression of the chimeric gene introduced.

1 22 1 1713 DNA Zea mays 1 aatcaatttc acacaggaaa cagctatgac catgattacgaattcgggcc cgggcgcgtg 60 atccggcggc ggcagcggcg gcggcggtgc aggcgggtgccgaggagatc gtgctgcagc 120 ccatcaagga gatctggggc agcgtcaagc tgccggggtccaagtcgctt tccaaccgga 180 tcctcctact cgccgccctg tccgagggga caacagtggttgataacctg ctgaacagtg 240 aggatgtcca ctacatgctc ggggccttga ggactcttggtctctctgtc gaagcggaca 300 aaggtcccaa aagagctgta cttgttggct ctggtggaaagttcccagtt gaggatgcta 360 aagaggaagt gcagctcttc ttggggaatg ctggaactgcaatgcggcca ttgacagcag 420 ctgttactgc tgctggtgga aatgcaactt acgtgcttgatggagtacca agaatgaggg 480 agagacccat tggcgacttg gttgtcggat tgaagcagcttggtgcagat gttgattgtt 540 tccttggcac tgactgccca cgtgttcgtg tcaatggaatcggagggcta cctggtggca 600 aggtcaagct gtctggctcc atcagcagtc agtacttgagtgccttgctg atggctgctc 660 ctttggctct tggggatgtg gagattgaaa tcattgataaattaatctcc attccgtacg 720 tcgaaatgac attgagattg atggaccgtt ttggtgtgaaagcagagcat tctgatagct 780 gggacagatt ctacattaag ggaggtcaaa aatacaagtcccctaaaaat gcctatgttg 840 aaggtgatgc ctcaagcgca agctatttct tggctggtgctgcaattact ggagggactg 900 tgactgtgga aggttgtggc accaccagtt tgcagggtgatgtgaagttt gctgaggtac 960 tggagatgat gggagcgaag gttacatgga ccgagactagcgtaactgtt actcccccac 1020 cgcgggagcc atttgggagg aaacacctca aggcgattgatgtcaacatc aacaagatgc 1080 ctgatgtcgc catgactctt gctgtggttg ccctctttgccgatggcccg acagccatca 1140 gagacgtggc ttcctggaga gtaaaggaga ccgagaggatggttgcgatc cggacggagc 1200 taaccaagct gggagcatct gttgaggaag ggccggactactgcatcatc acgccgccgg 1260 agaagctgaa cgtgacggcg atcgacacgt acgacgaccacaggatggcc atggccttct 1320 cccttgccgc ctgtgccgag gtccccgtca ccatccgggaccctgggtgc acccggaaga 1380 ccttccccga ctacttcgat gtgctgagca ctttcgtcaagaattaataa agcgtgcgat 1440 actaccacgc agcttgattg aagtgatagg cttgtgctgaggaaatacat ttcttttgtt 1500 ctgtttttct ctttcacggg attaagtttt gagtctgtaacgttagttgt ttgtagcaag 1560 tttctatttc ggatcttaag tttgtgcact gtaagccaaatttcatttca agagtggttc 1620 gttggaataa taagaataat aaattacgtt tcagtgaaaaaaaaaaaaaa aaaaaaaaaa 1680 aaaaaaaaaa aaaaaaaaaa aacccgggaa ttc 1713 21340 DNA Zea mays CDS (6)..(1337) 2 ccatg gcc ggc gcc gag gag atc gtgctg cag ccc atc aag gag atc tcc 50 Ala Gly Ala Glu Glu Ile Val Leu GlnPro Ile Lys Glu Ile Ser 1 5 10 15 ggc acc gtc aag ctg ccg ggg tcc aagtcg ctt tcc aac cgg atc ctc 98 Gly Thr Val Lys Leu Pro Gly Ser Lys SerLeu Ser Asn Arg Ile Leu 20 25 30 cta ctc gcc gcc ctg tcc gag ggg aca acagtg gtt gat aac ctg ctg 146 Leu Leu Ala Ala Leu Ser Glu Gly Thr Thr ValVal Asp Asn Leu Leu 35 40 45 aac agt gag gat gtc cac tac atg ctc ggg gccttg agg act ctt ggt 194 Asn Ser Glu Asp Val His Tyr Met Leu Gly Ala LeuArg Thr Leu Gly 50 55 60 ctc tct gtc gaa gcg gac aaa gct gcc aaa aga gctgta gtt gtt ggc 242 Leu Ser Val Glu Ala Asp Lys Ala Ala Lys Arg Ala ValVal Val Gly 65 70 75 tgt ggt gga aag ttc cca gtt gag gat gct aaa gag gaagtg cag ctc 290 Cys Gly Gly Lys Phe Pro Val Glu Asp Ala Lys Glu Glu ValGln Leu 80 85 90 95 ttc ttg ggg aat gct gga act gca atg cgg cca ttg acagca gct gtt 338 Phe Leu Gly Asn Ala Gly Thr Ala Met Arg Pro Leu Thr AlaAla Val 100 105 110 act gct gct ggt gga aat gca act tac gtg ctt gat ggagta cca aga 386 Thr Ala Ala Gly Gly Asn Ala Thr Tyr Val Leu Asp Gly ValPro Arg 115 120 125 atg agg gag aga ccc att ggc gac ttg gtt gtc gga ttgaag cag ctt 434 Met Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu LysGln Leu 130 135 140 ggt gca gat gtt gat tgt ttc ctt ggc act gac tgc ccacct gtt cgt 482 Gly Ala Asp Val Asp Cys Phe Leu Gly Thr Asp Cys Pro ProVal Arg 145 150 155 gtc aat gga atc gga ggg cta cct ggt ggc aag gtc aagctg tct ggc 530 Val Asn Gly Ile Gly Gly Leu Pro Gly Gly Lys Val Lys LeuSer Gly 160 165 170 175 tcc atc agc agt cag tac ttg agt gcc ttg ctg atggct gct cct ttg 578 Ser Ile Ser Ser Gln Tyr Leu Ser Ala Leu Leu Met AlaAla Pro Leu 180 185 190 gct ctt ggg gat gtg gag att gaa atc att gat aaatta atc tcc att 626 Ala Leu Gly Asp Val Glu Ile Glu Ile Ile Asp Lys LeuIle Ser Ile 195 200 205 ccg tac gtc gaa atg aca ttg aga ttg atg gag cgtttt ggt gtg aaa 674 Pro Tyr Val Glu Met Thr Leu Arg Leu Met Glu Arg PheGly Val Lys 210 215 220 gca gag cat tct gat agc tgg gac aga ttc tac attaag gga ggt caa 722 Ala Glu His Ser Asp Ser Trp Asp Arg Phe Tyr Ile LysGly Gly Gln 225 230 235 aaa tac aag tcc cct aaa aat gcc tat gtt gaa ggtgat gcc tca agc 770 Lys Tyr Lys Ser Pro Lys Asn Ala Tyr Val Glu Gly AspAla Ser Ser 240 245 250 255 gca agc tat ttc ttg gct ggt gct gca att actgga ggg act gtg act 818 Ala Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr GlyGly Thr Val Thr 260 265 270 gtg gaa ggt tgt ggc acc acc agt ttg cag ggtgat gtg aag ttt gct 866 Val Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly AspVal Lys Phe Ala 275 280 285 gag gta ctg gag atg atg gga gcg aag gtt acatgg acc gag act agc 914 Glu Val Leu Glu Met Met Gly Ala Lys Val Thr TrpThr Glu Thr Ser 290 295 300 gta act gtt act ggc cca ccg cgg gag cca tttggg agg aaa cac ctc 962 Val Thr Val Thr Gly Pro Pro Arg Glu Pro Phe GlyArg Lys His Leu 305 310 315 aag gcg att gat gtc aac atg aac aag atg cctgat gtc gcc atg act 1010 Lys Ala Ile Asp Val Asn Met Asn Lys Met Pro AspVal Ala Met Thr 320 325 330 335 ctt gct gtg gtt gcc ctc ttt gcc gat ggcccg aca gcc atc aga gac 1058 Leu Ala Val Val Ala Leu Phe Ala Asp Gly ProThr Ala Ile Arg Asp 340 345 350 gtg gct tcc tgg aga gta aag gag acc gagagg atg gtt gcg atc cgg 1106 Val Ala Ser Trp Arg Val Lys Glu Thr Glu ArgMet Val Ala Ile Arg 355 360 365 acg gag cta acc aag ctg gga gca tct gttgag gaa ggg ccg gac tac 1154 Thr Glu Leu Thr Lys Leu Gly Ala Ser Val GluGlu Gly Pro Asp Tyr 370 375 380 tgc atc atc acg ccg ccg gag aag ctg aacgtg acg gcg atc gac acg 1202 Cys Ile Ile Thr Pro Pro Glu Lys Leu Asn ValThr Ala Ile Asp Thr 385 390 395 tac gac gac cac agg atg gcc atg gcc ttctcc ctt gcc gcc tgt gcc 1250 Tyr Asp Asp His Arg Met Ala Met Ala Phe SerLeu Ala Ala Cys Ala 400 405 410 415 gag gtc ccc gtc acc atc cgg gac cctggg tgc acc cgg aag acc ttc 1298 Glu Val Pro Val Thr Ile Arg Asp Pro GlyCys Thr Arg Lys Thr Phe 420 425 430 ccc gac tac ttc gat gtg ctg agc actttc gtc aag aat taa 1340 Pro Asp Tyr Phe Asp Val Leu Ser Thr Phe Val LysAsn 435 440 3 444 PRT Zea mays 3 Ala Gly Ala Glu Glu Ile Val Leu Gln ProIle Lys Glu Ile Ser Gly 1 5 10 15 Thr Val Lys Leu Pro Gly Ser Lys SerLeu Ser Asn Arg Ile Leu Leu 20 25 30 Leu Ala Ala Leu Ser Glu Gly Thr ThrVal Val Asp Asn Leu Leu Asn 35 40 45 Ser Glu Asp Val His Tyr Met Leu GlyAla Leu Arg Thr Leu Gly Leu 50 55 60 Ser Val Glu Ala Asp Lys Ala Ala LysArg Ala Val Val Val Gly Cys 65 70 75 80 Gly Gly Lys Phe Pro Val Glu AspAla Lys Glu Glu Val Gln Leu Phe 85 90 95 Leu Gly Asn Ala Gly Thr Ala MetArg Pro Leu Thr Ala Ala Val Thr 100 105 110 Ala Ala Gly Gly Asn Ala ThrTyr Val Leu Asp Gly Val Pro Arg Met 115 120 125 Arg Glu Arg Pro Ile GlyAsp Leu Val Val Gly Leu Lys Gln Leu Gly 130 135 140 Ala Asp Val Asp CysPhe Leu Gly Thr Asp Cys Pro Pro Val Arg Val 145 150 155 160 Asn Gly IleGly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly Ser 165 170 175 Ile SerSer Gln Tyr Leu Ser Ala Leu Leu Met Ala Ala Pro Leu Ala 180 185 190 LeuGly Asp Val Glu Ile Glu Ile Ile Asp Lys Leu Ile Ser Ile Pro 195 200 205Tyr Val Glu Met Thr Leu Arg Leu Met Glu Arg Phe Gly Val Lys Ala 210 215220 Glu His Ser Asp Ser Trp Asp Arg Phe Tyr Ile Lys Gly Gly Gln Lys 225230 235 240 Tyr Lys Ser Pro Lys Asn Ala Tyr Val Glu Gly Asp Ala Ser SerAla 245 250 255 Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr ValThr Val 260 265 270 Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val LysPhe Ala Glu 275 280 285 Val Leu Glu Met Met Gly Ala Lys Val Thr Trp ThrGlu Thr Ser Val 290 295 300 Thr Val Thr Gly Pro Pro Arg Glu Pro Phe GlyArg Lys His Leu Lys 305 310 315 320 Ala Ile Asp Val Asn Met Asn Lys MetPro Asp Val Ala Met Thr Leu 325 330 335 Ala Val Val Ala Leu Phe Ala AspGly Pro Thr Ala Ile Arg Asp Val 340 345 350 Ala Ser Trp Arg Val Lys GluThr Glu Arg Met Val Ala Ile Arg Thr 355 360 365 Glu Leu Thr Lys Leu GlyAla Ser Val Glu Glu Gly Pro Asp Tyr Cys 370 375 380 Ile Ile Thr Pro ProGlu Lys Leu Asn Val Thr Ala Ile Asp Thr Tyr 385 390 395 400 Asp Asp HisArg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala Glu 405 410 415 Val ProVal Thr Ile Arg Asp Pro Gly Cys Thr Arg Lys Thr Phe Pro 420 425 430 AspTyr Phe Asp Val Leu Ser Thr Phe Val Lys Asn 435 440 4 1340 DNA Zea maysCDS (6)..(1337) 4 ccatg gcc ggc gcc gag gag atc gtg ctg cag ccc atc aaggag atc tcc 50 Ala Gly Ala Glu Glu Ile Val Leu Gln Pro Ile Lys Glu IleSer 1 5 10 15 ggc acc gtc aag ctg ccg ggg tcc aag tcg ctt tcc aac cggatc ctc 98 Gly Thr Val Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg IleLeu 20 25 30 cta ctc gcc gcc ctg tcc gag ggg aca aca gtg gtt gat aac ctgctg 146 Leu Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu35 40 45 aac agt gag gat gtc cac tac atg ctc ggg gcc ttg agg act ctt ggt194 Asn Ser Glu Asp Val His Tyr Met Leu Gly Ala Leu Arg Thr Leu Gly 5055 60 ctc tct gtc gaa gcg gac aaa gct gcc aaa aga gct gta gtt gtt ggc242 Leu Ser Val Glu Ala Asp Lys Ala Ala Lys Arg Ala Val Val Val Gly 6570 75 tgt ggt gga aag ttc cca gtt gag gat gct aaa gag gaa gtg cag ctc290 Cys Gly Gly Lys Phe Pro Val Glu Asp Ala Lys Glu Glu Val Gln Leu 8085 90 95 ttc ttg ggg aat gct gga atc gca atg cgg tcc ttg aca gca gct gtt338 Phe Leu Gly Asn Ala Gly Ile Ala Met Arg Ser Leu Thr Ala Ala Val 100105 110 act gct gct ggt gga aat gca act tac gtg ctt gat gga gta cca aga386 Thr Ala Ala Gly Gly Asn Ala Thr Tyr Val Leu Asp Gly Val Pro Arg 115120 125 atg agg gag aga ccc att ggc gac ttg gtt gtc gga ttg aag cag ctt434 Met Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu 130135 140 ggt gca gat gtt gat tgt ttc ctt ggc act gac tgc cca cct gtt cgt482 Gly Ala Asp Val Asp Cys Phe Leu Gly Thr Asp Cys Pro Pro Val Arg 145150 155 gtc aat gga atc gga ggg cta cct ggt ggc aag gtc aag ctg tct ggc530 Val Asn Gly Ile Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly 160165 170 175 tcc atc agc agt cag tac ttg agt gcc ttg ctg atg gct gct cctttg 578 Ser Ile Ser Ser Gln Tyr Leu Ser Ala Leu Leu Met Ala Ala Pro Leu180 185 190 gct ctt ggg gat gtg gag att gaa atc att gat aaa tta atc tccatt 626 Ala Leu Gly Asp Val Glu Ile Glu Ile Ile Asp Lys Leu Ile Ser Ile195 200 205 ccg tac gtc gaa atg aca ttg aga ttg atg gag cgt ttt ggt gtgaaa 674 Pro Tyr Val Glu Met Thr Leu Arg Leu Met Glu Arg Phe Gly Val Lys210 215 220 gca gag cat tct gat agc tgg gac aga ttc tac att aag gga ggtcaa 722 Ala Glu His Ser Asp Ser Trp Asp Arg Phe Tyr Ile Lys Gly Gly Gln225 230 235 aaa tac aag tcc cct aaa aat gcc tat gtt gaa ggt gat gcc tcaagc 770 Lys Tyr Lys Ser Pro Lys Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser240 245 250 255 gca agc tat ttc ttg gct ggt gct gca att act gga ggg actgtg act 818 Ala Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr ValThr 260 265 270 gtg gaa ggt tgt ggc acc acc agt ttg cag ggt gat gtg aagttt gct 866 Val Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys PheAla 275 280 285 gag gta ctg gag atg atg gga gcg aag gtt aca tgg acc gagact agc 914 Glu Val Leu Glu Met Met Gly Ala Lys Val Thr Trp Thr Glu ThrSer 290 295 300 gta act gtt act ggc cca ccg cgg gag cca ttt ggg agg aaacac ctc 962 Val Thr Val Thr Gly Pro Pro Arg Glu Pro Phe Gly Arg Lys HisLeu 305 310 315 aag gcg att gat gtc aac atg aac aag atg cct gat gtc gccatg act 1010 Lys Ala Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala MetThr 320 325 330 335 ctt gct gtg gtt gcc ctc ttt gcc gat ggc ccg aca gccatc aga gac 1058 Leu Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Ala IleArg Asp 340 345 350 gtg gct tcc tgg aga gta aag gag acc gag agg atg gttgcg atc cgg 1106 Val Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Val AlaIle Arg 355 360 365 acg gag cta acc aag ctg gga gca tct gtt gag gaa gggccg gac tac 1154 Thr Glu Leu Thr Lys Leu Gly Ala Ser Val Glu Glu Gly ProAsp Tyr 370 375 380 tgc atc atc acg ccg ccg gag aag ctg aac gtg acg gcgatc gac acg 1202 Cys Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Ala IleAsp Thr 385 390 395 tac gac gac cac agg atg gcg atg gcc ttc tcc ctt gccgcc tgt gcc 1250 Tyr Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala AlaCys Ala 400 405 410 415 gag gtc ccc gtc acc atc cgg gac cct ggg tgc acccgg aag acc ttc 1298 Glu Val Pro Val Thr Ile Arg Asp Pro Gly Cys Thr ArgLys Thr Phe 420 425 430 ccc gac tac ttc gat gtg ctg agc act ttc gtc aagaat taa 1340 Pro Asp Tyr Phe Asp Val Leu Ser Thr Phe Val Lys Asn 435 4405 444 PRT Zea mays 5 Ala Gly Ala Glu Glu Ile Val Leu Gln Pro Ile Lys GluIle Ser Gly 1 5 10 15 Thr Val Lys Leu Pro Gly Ser Lys Ser Leu Ser AsnArg Ile Leu Leu 20 25 30 Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val AspAsn Leu Leu Asn 35 40 45 Ser Glu Asp Val His Tyr Met Leu Gly Ala Leu ArgThr Leu Gly Leu 50 55 60 Ser Val Glu Ala Asp Lys Ala Ala Lys Arg Ala ValVal Val Gly Cys 65 70 75 80 Gly Gly Lys Phe Pro Val Glu Asp Ala Lys GluGlu Val Gln Leu Phe 85 90 95 Leu Gly Asn Ala Gly Ile Ala Met Arg Ser LeuThr Ala Ala Val Thr 100 105 110 Ala Ala Gly Gly Asn Ala Thr Tyr Val LeuAsp Gly Val Pro Arg Met 115 120 125 Arg Glu Arg Pro Ile Gly Asp Leu ValVal Gly Leu Lys Gln Leu Gly 130 135 140 Ala Asp Val Asp Cys Phe Leu GlyThr Asp Cys Pro Pro Val Arg Val 145 150 155 160 Asn Gly Ile Gly Gly LeuPro Gly Gly Lys Val Lys Leu Ser Gly Ser 165 170 175 Ile Ser Ser Gln TyrLeu Ser Ala Leu Leu Met Ala Ala Pro Leu Ala 180 185 190 Leu Gly Asp ValGlu Ile Glu Ile Ile Asp Lys Leu Ile Ser Ile Pro 195 200 205 Tyr Val GluMet Thr Leu Arg Leu Met Glu Arg Phe Gly Val Lys Ala 210 215 220 Glu HisSer Asp Ser Trp Asp Arg Phe Tyr Ile Lys Gly Gly Gln Lys 225 230 235 240Tyr Lys Ser Pro Lys Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser Ala 245 250255 Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr Val Thr Val 260265 270 Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe Ala Glu275 280 285 Val Leu Glu Met Met Gly Ala Lys Val Thr Trp Thr Glu Thr SerVal 290 295 300 Thr Val Thr Gly Pro Pro Arg Glu Pro Phe Gly Arg Lys HisLeu Lys 305 310 315 320 Ala Ile Asp Val Asn Met Asn Lys Met Pro Asp ValAla Met Thr Leu 325 330 335 Ala Val Val Ala Leu Phe Ala Asp Gly Pro ThrAla Ile Arg Asp Val 340 345 350 Ala Ser Trp Arg Val Lys Glu Thr Glu ArgMet Val Ala Ile Arg Thr 355 360 365 Glu Leu Thr Lys Leu Gly Ala Ser ValGlu Glu Gly Pro Asp Tyr Cys 370 375 380 Ile Ile Thr Pro Pro Glu Lys LeuAsn Val Thr Ala Ile Asp Thr Tyr 385 390 395 400 Asp Asp His Arg Met AlaMet Ala Phe Ser Leu Ala Ala Cys Ala Glu 405 410 415 Val Pro Val Thr IleArg Asp Pro Gly Cys Thr Arg Lys Thr Phe Pro 420 425 430 Asp Tyr Phe AspVal Leu Ser Thr Phe Val Lys Asn 435 440 6 418 DNA Zea mays 6 tgaggtacgattcttcgatc ctctttgatt ttcctggaaa tattttttcg gtgatcgtga 60 aactactggaatcgctcgat aggtggtacg aaattaggcg agattagttt ctattcttgg 120 ccattatcttgtttcttcgc cgaatgatct tccgtataaa gattttaggt tagagatgaa 180 tcgtatagctagatttcatc accagatagt ttctttgtct agaatctctg aaattctcga 240 tagttttcacatgtgtaaat agattgttct tattcggcga ttgttgatta gggttttgat 300 tttcttgattatgcgattgc aattagggat tttctttggt tttgtgttga tcttacgata 360 cattcctgcaattgaatacg tatggatcta aatcttgtta atttgttgaa cagatccc 418 7 494 DNA Zeamays 7 ctcaggcgaa gaacaggtat gatttgtttg taattagatc aggggtttag gtctttccat60 tactttttaa tgttttttct gttactgtct ccgcgatctg attttacgac aatagagttt 120cgggttttgt cccattccag tttgaaaata aacgtccgtc ttttaagttt gctggatcga 180taaacctgtg aagattgagt ctagtcgatt tattggatga tccattcttc atcgtttttt 240tcttgcttcg aagttctgta taaccagatt tgtctgtgtg cgattgtcat tacctagccg 300tgtatcgaga actagggttt tcgagtcaat tttgcccctt ttggttatat ctggttcgat 360aacgattcat ctggattagg gttttaagtg gtgacgttta gtattccaat ttcttcaaaa 420tttagttatg gataatgaaa atcccgaatt gactgttcaa tttcttgtta aatgcgcaga 480tcccgggatc tgcg 494 8 20 DNA Zea mays 8 gctctgctca tgtctgctcc 20 9 20DNA Zea mays 9 gcccgccctt gacaaagaaa 20 10 10 DNA Zea mays 10 aattcccggg10 11 38 DNA Zea mays 11 aattaagctc tagagtcgac ctgcaggcat gcaagctt 38 1241 DNA Zea mays 12 gagccgagct ccatggccgg cgccgaggag atcgtgctgc a 41 1337 DNA Zea mays 13 gcacgatctc ctcggcgccg gccatggagc tcggctc 37 14 25 DNAZea mays 14 ccacaggatg gcgatggcct tctcc 25 15 33 DNA Zea mays 15gaatgctgga atcgcaatgc ggccattgac agc 33 16 33 DNA Zea mays 16 gaatgctggaactgcaatgc ggtccttgac agc 33 17 34 DNA Zea mays 17 cttggggaat gctgccatcgcaatgcggcc attg 34 18 36 DNA Zea mays 18 ggggaatgct ggaatcgcaatgcggtcctt gacagc 36 19 20 DNA Zea mays 19 taatttgttg aacagatccc 20 2018 DNA Zea mays 20 taaacaactt gtctaggg 18 21 18 DNA Zea mays 21cagatcccgg gatctgcg 18 22 20 DNA Zea mays 22 gcgtctaggg ccctagacgc 20

1. An isolated DNA sequence capable of serving as a regulatory element in a chimeric gene which can be used for the transformation of plants and allowing the expression of the product of translation of the chimeric gene in particular in the regions of the plant undergoing rapid growth, characterized in that it comprises at least one intron such as the first intron (intron 1) of the noncoding 5′ region of a plant histone gene.
 2. DNA sequence according to claim 1, characterized in that histone intron comes from a plant histone gene of the “H3.3-like” type.
 3. DNA sequence according to either of claims 1 and 2, characterized in that histone intron 1 comes from a dicotyledonous plant.
 4. DNA sequence according to claim 3, characterized in that histone intron 1 comes from Arabidopsis thaliana.
 5. DNA sequence according to either of claims 1 and 2, characterized in that histone intron 1 comes from a monocotyledonous plant.
 6. DNA sequence according to claim 5, characterized in that histone intron 1 comes from Zea mays.
 7. DNA sequence according to any one of claims 1 to 6, characterized in that intron 1 is oriented, in the direction of transcription of the chimeric gene, in a direct or reversed manner relative to its initial orientation in the direction of transcription of the gene from which it is derived.
 8. DNA sequence according to any one of claims 1 to 7, characterized in that the regulatory element comprises two introns 1, identical or different, which are combined.
 9. Chimeric gene for the transformation of plants comprising at least, in the direction of transcription, one regulatory element comprising a promoter sequence, a sequence of a herbicide tolerance gene and a regulatory element, characterized in that the regulatory element comprises, in addition, an intron 1 according to any one of claims 1 to
 8. 10. Chimeric gene according to claim 9, characterized in that the promoter sequence comes from a promoter of a plant histone gene.
 11. Chimeric gene according to either of claims 9 and 10, characterized in that the promoter zone comes from the same plant histone gene as intron
 1. 12. Chimeric gene according to any one of claims 9 to 11, characterized in that the promoter sequence comprises a promoter for a duplicated plant histone.
 13. Chimeric gene according to any one of claims 9 to 12, characterized in that the promoter sequence contains at least one promoter of a plant histone gene combined with a different promoter derived from a gene which can be naturally expressed in plants.
 14. Chimeric gene according to one of claims 9 to 13, characterized in that the coding gene makes it possible to confer on plants an enhanced tolerance to a herbicide.
 15. Chimeric gene according to claim 14, characterized in that the herbicide tolerance gene is fused with a DNA sequence encoding a signal peptide allowing the accumulation of the product of translation of the herbicide tolerance gene in a subcellular compartment.
 16. Chimeric gene according to claim 15, characterized in that the signal peptide zone allows the accumulation of the product of translation of the herbicide tolerance gene in the plastid compartment.
 17. Chimeric gene according to claim 16, characterized in that the signal peptide sequence comprises, in the direction of transcription, at least one signal peptide sequence of a plant gene encoding a signal peptide directing transport of a polypeptide to a plastid, optionally a portion of the sequence of the mature N-terminal part of a plant gene produced when the first signal peptide is cleaved by proteolytic enzymes, and then optionally a second signal peptide of a plant gene encoding a signal peptide directing transport of the polypeptide to a sub-compartment of the plastid.
 18. Chimeric gene according to any one of claims 9 to 17, characterized in that the herbicide tolerance gene encodes an enzyme which is active towards herbicides whose target is EPSPS.
 19. Chimeric gene according to claim 18, characterized in that the herbicide tolerance gene encodes an enzyme which is active towards glyphosate.
 20. Vector for the transformation of plants, characterized in that it comprises a chimeric gene according to any one of claims 9 to
 19. 21. Strain of Agrobacterium sp., characterized in that it contains a vector according to claim
 20. 22. Transformed plant cells, characterized in that it contains a chimeric gene according to any one of claims 9 to
 19. 23. Transformed plant or plant portion obtained from a cell according to claim
 22. 24. Process for the construction of a chimeric gene according to any one of claims 9 to 23, which comprises isolating an intron 1 from a plant histons gene as defined in any one of claims 1 to 8, a promoter sequence, a signal peptide, and at least one transgene, and assembling them, in that order in the direction of transcription of the transgene. 